Members of the nucleobase/ascorbate transporter (NAT) gene family transport nucleobases in all kingdoms of live and vitamin C in mammals. In humans, vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant scavenger of free radicals and as a cofactor in many enzymatic reactions. Transport of nucleobases is implicated in crucial processes such as DNA and RNA synthesis, cell signaling, and metabolic regulation. In addition, the cellular delivery of nucleobases has gained special interest in therapeutic applications as nucleobase analogs are currently used in the treatment of solid tumors, lymphoproliferative diseases, viral infections such as hepatitis and AIDS, and some inflammatory diseases, e.g., Crohn's disease. Despite the importance of NATs in health, disease, and pharmacotherapy, detailed information about their transport mechanism, which is crucial to exploit their potential as target for drugs with high efficacy, is limited. In this multple PD/PI proposal we seek to understand mechanistic commonalities and differences among members of the NAT family. Building on our recent exciting identification and crystallization of a bacterial NAT homolog (PaaTCp) at 2.85 resolution that transports nucleobases and vitamin C in H+ and Na+-dependent fashion, respectively, this project is designed to elucidate basic mechanisms of substrate recognition and translocation in both bacterial and human NAT family members. We propose the following Specific Aims: (1) to identify the substrate and drug binding site(s). The goal is to co-crystallize PaaTCp with its substrates (purines and vitamin C) and nucleobase analogs and then use the structures as a guide to functionally validate the substrate and drug binding sites by mutational studies in conjunction with radiotracer binding; (2) to develop a model of transport for PaaTCp. The goal is to obtain a quantitative understanding of H+- and Na+-dependent substrate transport, including the identification of the H+ and Na+ sites and the elucidation of the stoichiometry of the potential ion (H+ and Na+) coupling mechanism, and to describe precisely the kinetics of transport; (3) to illustrate conformational changes associated with (co)substrate translocation and how drugs affect these transitions. The goal is to crystallize PaaTCp in outward- and inward-facing conformations, and to use crosslinking and cysteine accessibility assays to validate the structures, or when structures with alternate conformations are not attainable, to deduce conformational changes; (4) to establish the relevance of our structural and functional findings in PaaTCp to understanding the function of the human SVCTs by exploring the key elements of substrate binding, and its coupling to the ion motive force to develop a general applicable mechanistic model of function for proteins with PaaTCp fold.